Synovium-derived stromal cell-induced osteoclastogenesis: a potential osteoarthritis trigger

none7noPurpose: To shed light on the idea that mesenchymal stem/stromal cells (MSCs) recruited in synovium (SM) (i.e. Synovium-Derived Stromal Cells, SDSCs) could be involved in Osteoarthritis (OA) pathophysiology. Attention was also paid to a further stromal cell type with a peculiar ultrastructure called telocytes (TCs), whose role is far from clarified. Methods: In the present in vitro study, we compared SDSCs isolated from healthy and OA subjects in terms of phenotype, morphology and differentiation potential as well as in their capability to activate normal Peripheral Blood Mononuclear Cells (PBMCs). Histological, immunohistochemical and ultrastructural analyses were integrated by qRT-PCR and functional resorbing assays. Results: Our data demonstrated that both SDSC populations stimulated the formation of osteoclasts from PBMCs: the osteoclast-like cells generated by healthy-SDSCs via transwell co-cultures were inactive, while OA-derived SDSCs have a much greater effectiveness. Moreover, the presence of TCs was more evident in cultures obtained from OA subjects and suggests a possible involvement of these cells in OA. Conclusions: Osteoclastogenic differentiation capability of PBMCs from OA subjects, also induced by B synoviocytes has been already documented. Here we hypothesized that SDSCs, generally considered for their regenerative potential in cartilage lesions, have also a role in the onset/maintenance of OA. Clinical relevance: Our observations may represent an interesting opportunity for the development of a holistic approach for OA treatment, that considers the multifaceted capability of MSCs in relation to the environment.embargoed_20210517Dicarlo, Manuela; Teti, Gabriella; Cerqueni, Giorgia; Iezzi, Iolanda; Gigante, Antonio; Falconi, Mirella; Mattioli-Belmonte, MonicaDicarlo, Manuela; Teti, Gabriella; Cerqueni, Giorgia; Iezzi, Iolanda; Gigante, Antonio; Falconi, Mirella; Mattioli-Belmonte, Monic

Biofabrication of bundles of poly(lactic acid)-collagen blends mimicking the fascicles of the human Achille tendon

Electrospinning is a promising technique for the production of scaffolds aimed at the regeneration of soft tissues. The aim of this work was to develop electrospun bundles mimicking the architecture and mechanical properties of the fascicles of the human Achille tendon. Two different blends of poly(L-lactic acid) (PLLA) and collagen (Coll) were tested, PLLA/Coll-75/25 and PLLA/Coll-50/50, and compared with bundles of pure PLLA. First, a complete physico-chemical characterization was performed on non-woven mats made of randomly arranged fibers. The presence of collagen in the fibers was assessed by thermogravimetric analysis, differential scanning calorimetry and water contact angle measurements. The collagen release in phosphate buffer solution (PBS) was evaluated for 14 days: results showed that collagen loss was about 50% for PLLA/Coll-75/25 and 70% for PLLA/Coll-50/50. In the bundles, the individual fibers had a diameter of 0.48 ±0.14 μm (PLLA), 0.31 ±0.09 μm (PLLA/Coll-75/25), 0.33 ±0.08 μm (PLLA/Coll-50/50), whereas bundle diameter was in the range 300-500 μm for all samples. Monotonic tensile tests were performed to measure the mechanical properties of PLLA bundles (as-spun) and of PLLA/Coll-75/25 and PLLA/Coll-50/50 bundles (as-spun, and after 48 h, 7 days and 14 days in PBS). The most promising material was the PLLA/Coll-75/25 blend with a Young modulus of 98.6 ±12.4 MPa (as-spun) and 205.1 ±73.0 MPa (after 14 days in PBS). Its failure stress was 14.2 ±0.7 MPa (as-spun) and 6.8 ±0.6 MPa (after 14 days in PBS). Pure PLLA withstood slightly lower stress than the PLLA/Coll-75/25 while PLLA/Coll-50/50 had a brittle behavior. Human-derived tenocytes were used for cellular tests. A good cell adhesion and viability after 14 day culture was observed. This study has therefore demonstrated the feasibility of fabricating electrospun bundles with multiscale structure and mechanical properties similar to the human tendon

Cobalt chloride supplementation differently affects human mesenchymal stem cells isolated from dental pulp, umbilical cord and adipose tissues in their chondrogenic potential

Articular cartilage is an avascular tissue without innervations, characterized by low cell density and abundant extracellular matrix (ECM). These characteristics leave articular cartilage with very limited capacity of repair and regeneration. Multipotent stem/stromal cells (MSC) are considered promising for cartilage tissue engineering. Stem cells are resided in a special microenvironment known as the stem-cell niche, characterized by the presence of low oxygen concentration. Previous studies have reported that hypoxic conditions could enhance the chondrogenic differentiation of mesenchymal stem cells in the presence of an inductive medium. Cobalt chloride (CoCl2) imitates hypoxia in vitro by preventing hypoxia-inducible factor-alpha (HIF-a) from being destroyed by oxygen. However, the long-term hypoxic culture of stem cells is difficult and requires special attention to avoid cell death due to cobalt treatment. In this study we investigated if CoCl2 affected MSCs isolated from dental pulp, umbilical cord and adipose tissue in their potential to differentiate toward the chondrogenic phenotype. Cells were treated with concentrations of CoCl2 ranging from 50 to 400 uM. Cell proliferation, mRNA expression of stem-cell marker and chondrogenic associated genes were analyzed by RT-PCR and Real-time PCR. The results showed that the CoCl2 supplementation had no effect on the proliferation of all the three type of cells analyzed, while the up-regulation of chondrogenic markers such as aggrecan, sox9, and type II collagen, was dependent on the cellular source. This study shows that hypoxia induced by CoCl2 treatment can differently influence the behavior of MSCs of different sources in their chondrogenic potential. These findings should be taken into consideration in the treatment of cartilage repair and regeneration based on stem cell therapies

Bone regeneration strategies in the elderly: the role of ageing and replicative senescence in periosteal-derived stem cells

Periosteum contains resident progenitor cells (PDPCs) representing an attractive alternative source of mesenchymal stem cells (MSCs) for skeletal tissue engineering approaches based on cell recruitment (1). Increased in life expectancy point out the necessity for customized strategies to restore bone loss due to trauma and/or disease in elderly. Aim of the present research was the evaluation of the ageing impact on PDPCs isolated from differently aged subjects. Moreover, since long-term culture could lead MSCs to senescence, the effects of culture expansion method on young PDPC through sequential serial passages were examined. Age-related increase of p53 expression and impairment in proliferating capacity were observed; those findings were strictly related to nitric oxide (NO) release. Moreover, qRT-PCR analysis showed a greater expression of genes involved in bone remodelling in elderly donors. As far as replicative in vitro expansion was concerned, we observed that later PDPC passages exhibited the typical “replicative senescence” features (i.e. flattened and enlarged morphology, prolonged population doubling time and increased SA-βgal activity). In these cells, p16 rather than p53 seemed to be involved in senescence processes. Similarly to the elderly, the decrease in proliferating ability of in vitro senescent PDPCs was concomitant with a higher NO production, and the changes in the expression of genes involved in bone resorption and RANKL/OPG ratio were superimposable. Interestingly, the relationship between NO release and ageing could represent a cutting edge “replicative senescence index” as emerged by our System Biology approach. In conclusion, our findings suggest that in vivo cell ageing and in vitro subculturing must be taken into account when testing regenerative tissue strategies that use progenitor cells. Indeed, cells (e.g. MSCs and PDPCs) from the earliest subculture passages could be useful to validate any bone tissue engineering strategies, whilst the later ones could be used to test in vitro scaffolds for regenerative medicine approaches in elderly.This work was supported by grants from MIUR (Project PRIN 2010, MIND-2010J8RYS7)

Aging of periosteal-derived stem cells during expansion: an alternative tool for a customized bone regenerative strategy

Increased in life expectancy points out the necessity for tailored strategies to restore bone loss due to trauma and/or disease in elderly. Moreover, there is a compelling need for improved cell systems to test scaffolds interfacing with an “aging” tissue. For skeletal tissue regeneration, periosteal-derived stem cells (PDPCs) could represent an easily recruited source of Mesenchymal stromal cells (MSCs) [1,2]. This study investigated the effects of long-term in vitro expansion on the stability and function of PDPCs, since extensive culture expansion is usually performed to obtain clinically relevant cell numbers, but its impact on cell behaviour is still unclear. An integrated approach based on flow cytometry, ultrastructural and quantitative Real time PCR (qRT-PCR) analyses was adopted. Senescent cell data were compared with those of cells isolated from differently aged subjects. Both replicative-senescent PDPCs and cells isolated from old donors were permanently blocked in G1 phase of cell cycle, through a pathway that seemed to involve nitric oxide (NO) production and the expression of tumour suppressor proteins p16 or p53, respectively. Changes in the expression of MSC surface markers were detected in PDPCs during subculturing, whilst it was superimposable in young and aged PDPCs. Cytofluorimetric analysis of the physical parameters (i.e. FSC and SSC) showed a trend toward an increase in cell dimension and internal complexity in both populations analysed. This data was consistent with morphological observation that also evidenced similar alterations in mitochondrial shape. In addition, an intense autophagic activity in early passage PDPCs was observed, whilst in the late passages cells had a robust protein synthesis activity that could be related with “senescence-associated secretory phenotype” (SASP). In conclusion, the morphofunctional similarities detected in replicative-senescent and aged PDPCs suggest that their long-term expansion could be a reproducible and useful tool to mimic in vivo ageing

Quercetin exposure suppresses the inflammatory pathway in intestinal organoids from winnie mice

Inflammatory bowel diseases (IBDs) are chronic and relapsing immune disorders that result, or possibly originate, from epithelial barrier defects. Intestinal organoids are a new reliable tool to investigate epithelial response in models of chronic inflammation. We produced organoids from the ulcerative colitis murine model Winnie to explore if the chronic inflammatory features observed in the parental intestine were preserved by the organoids. Furthermore, we investigated if quercetin administration to in vitro cultured organoids could suppress LPS-induced inflammation in wild-type organoids (WT-organoids) and spontaneous inflammation in ulcerative colitis organoids (UC-organoids). Our data demonstrate that small intestinal organoids obtained from Winnie mice retain the chronic intestinal inflammatory features characteristic of the parental tissue. Quercetin administration was able to suppress inflammation both in UC-organoids and in LPS-treated WT-organoids. Altogether, our data demonstrate that UC-organoids are a reliable experimental system for investigating chronic intestinal inflammation and pharmacological responses

Age-related changes in human periosteal derived stem cells: a matter for effective bone regeneration strategies

Possible age-related changes in Mesenchymal Stem Cells (MSCs) are of great inter- est in view of their use for regenerative medicine approaches also in the elderly. Con- sidering the primary role of periosteum in bone biology and to acquire data for a cell- based therapy stimulating graft osseointegration, we tried to identify specific aging markers or pattern of expression in human periosteal precursor cells (PDPCs). To this aim periosteal tissue was obtained from differently aged healthy subjects, gender matched with a mean age of 16, 28, 63 and 92 years. Immunohistochemical detection of Ki67 and p53, Nitric Oxide (NO) production and qRT- PCR of a selected gene pan- el for early osteoblastic differentiation (bmp2 and runx2) and bone remodelling (IL-6, RANKL and OPG) were evaluated. Our data evidenced that both Ki67 and p53 rep- resent striking markers of cell-cycle arrest in these cells and that their expression cor- relates with NO production. In addition, age affects genes involved in bone remodel- ling, with a significant increase in IL-6 mRNA expression as well as in RANKL/OPG ratio. As far as NO release is concerned, our data showed higher levels in PDPCs iso- lated from the elderly and a good correlation with the immunohistochemical analysis Moreover, mathematical modelling, in silico simulations and biochemical experiments were combined to investigate about possible underlying quantitative correlations. A clear one-term exponential relationship emerged from a comparison of involved marker trends against age of donors concerning measured NO concentration / Ki67 ratio. This analytical approach confirmed Ki67 as a senescence marker to be focused on. We believe that this study, taking into account age-related changes in human PDPCs, opens up new regenerative medicine strategies for aged bone and/or bone metabolic diseases

A Bronze-Tomato Enriched Diet Affects the Intestinal Microbiome under Homeostatic and Inflammatory Conditions

Inflammatory bowel diseases (IBD) are debilitating chronic inflammatory disorders that develop as a result of a defective immune response toward intestinal bacteria. Intestinal dysbiosis is associated with the onset of IBD and has been reported to persist even in patients in deep remission. We investigated the possibility of a dietary-induced switch to the gut microbiota composition using Winnie mice as a model of spontaneous ulcerative colitis and chow enriched with 1% Bronze tomato. We used the near isogenic tomato line strategy to investigate the effects of a diet enriched in polyphenols administered to mild but established chronic intestinal inflammation. The Bronze-enriched chow administered for two weeks was not able to produce any macroscopic effect on the IBD symptoms, although, at molecular level there was a significant induction of anti-inflammatory genes and intracellular staining of T cells revealed a mild decrease in IL17A and IFNγ production. Analysis of the microbial composition revealed that two weeks of Bronze enriched diet was sufficient to perturb the microbial composition of Winnie and control mice, suggesting that polyphenol-enriched diets may create unfavorable conditions for distinct bacterial species. In conclusion, dietary regimes enriched in polyphenols may efficiently support IBD remission affecting the intestinal dysbiosis

A Specific Mutation in Muc2 Determines Early Dysbiosis in Colitis-Prone Winnie Mice

BACKGROUND: Inflammatory bowel disease (IBD), including Crohn disease (CD) and ulcerative colitis (UC), is a multifactorial disorder characterized by chronic inflammation and altered gut barrier function. Dysbiosis, a condition defined by dysregulation of the gut microbiome, has been reported in patients with IBD and in experimental models of colitis. Although several factors have been implicated in directly affecting gut microbial composition, the genetic determinants impacting intestinal dysbiosis in IBD remain relatively unknown. METHODS: We compared the microbiome of normal, uninflamed wild-type (WT) mice with that of a murine model of UC (ie, Winnie strain). Winnie mice possess a missense mutation in Muc2 that manifests in altered mucus production as early as 4 weeks of age, with ensuing colonic inflammation. To better address the potential role of mutant Muc2 in promoting dysbiosis in Winnie mice, we evaluated homozygous mutant mice (Winnie-/-) with their WT littermates that, after weaning from common mothers, were caged separately according to genotype. Histologic and inflammatory status were assessed over time, along with changes in their respective microbiome compositions. RESULTS: Dysbiosis in Winnie mice was already established at 4 weeks of age, before histologic evidence of gut inflammatory changes, in which microbial communities diverged from that derived from their mothers. Furthermore, dysbiosis persisted until 12 weeks of age, with peak differences in microbiome composition observed between Winnie and WT mice at 8 weeks of age. The relative abundance of Bacteroidetes was greater in Winnie compared with WT mice. Verrucomicrobia was detected at the highest relative levels in 4-week-old Winnie mice; in particular, Akkermansia muciniphila was among the most abundant species found at 4 weeks of age. CONCLUSIONS: Our results demonstrate that mutant genetic determinants involved in the complex regulation of intestinal homeostasis, such as that observed in Winnie mice, are able to promote early gut dysbiosis that is independent from maternal microbial transfer, including breastfeeding. Our data provide evidence for intestinal dysbiosis attributed to a Muc2-driven mucus defect that leads to colonic inflammation and may represent an important target for the design of future interventional studies